Skin Treatment

ABSTRACT

The present invention provides a method for determining the predisposition of an individual to a skin condition comprising identifying the profilaggrin alleles present in the genome of an ex vivo sample taken from the individual. Skin conditions include the ability of an individual to produce Natural Moisturising Factors (NMF), dry skin and/or predisposition to detergent-induced erythema. Typically profilaggrin alleles are identified by determining the number of encoded filaggrin repeats. Methods of the invention are particularly useful for grouping individuals for the purposes of participation in clinical trials or for matching an individual to a cosmetic preparation.

This is a Continuation of co-pending U.S. patent application Ser. No.10/140,694 filed May 8, 2002, which claims priority under 35 U.S.C. §119to United Kingdom Application No. 0111324.0 filed May 9, 2001; all ofwhich are incorporated herein in their entirety, by reference.

FIELD OF THE INVENTION

The present invention relates to a method of allele identification. Moreparticularly the present invention relates to a method for identifyingthe profilaggrin alleles present in the genome of an individual,knowledge of which can be used to determine the individual'spredisposition to skin conditions.

BACKGROUND OF THE INVENTION

During epidermal differentiation, keratinocytes undergo a well definedseries of morphological and biochemical changes in which activelyproliferating basal cells differentiate stepwise through the spinous andgranular cell layers to eventually form the anuclear squamescharacteristic of the protective stratum corneum at the skin surface(Presland et al (1992) J Biol Chem, 267(33), 23772-23781). Eachepidermal layer is characterised by the expression of specificbiochemical markers of which the keratin intermediate filament proteins,K5/K14 and K1/K10, predominate in the basal and spinous layers,respectively (Presland et al (1992) J Biol Chem, 267(33), 23772-23781).

Granular cells are characterised by the expression of profilaggrin. Theprofilaggrin gene encodes a high molecular weight phosphorylatedpolyprotein, composed of a number of related but nonidentical filaggrinrepeats. Peptide mapping and sequencing studies have revealed thatfilaggrin units are separated by short linker peptides which are removedduring proteolytic processing (Presland et al (1992) J Biol Chem,267(33), 23772-23781). Like its rodent counterparts, the coding regionof the human profilaggrin gene contains no introns within the repetitiveportion of the coding region.

Phosphorylated profilaggrin is non-functional and accumulates asF-keratohyalin granules late in epidermal differentiation (Gan et al(1990) Biochemistry, 29, 9432-9440). During the transition from thegranular to the terminally differentiated cornified cell, profilaggrinis dephosphorylated and proteolytically processed to yield filaggrinmonomers. Filaggrin participates in the aggregation of keratinintermediate filaments into the dense macrofibrils characteristic of thestratum corneum (Presland et al (1992) J Biol Chem, 267(33),23772-23781). Profilaggrin may also play a role in maintaining epidermalhydration through the degradation of filaggrin to free amino acids(Presland et al (1992) J Biol Chem, 267(33), 23772-23781). The freeamino acids form part of the Natural Moisturising Factors (NMF) ofstratum corneum. NMF maintains the hydration of the skin and hence itscondition.

The profilaggrin gene is located on Chromosome 1q21 as part of thecluster of genes known as the Epidermal Differentiation Complex (EDC)(Mishke et al (1996) SID 106(5): 989-992). Many of these genes encodeproducts which are believed to contribute to stratum corneum structureand function. The profilaggrin gene has been reported to be polymorphicin size due to allelic differences in the number of filaggrin repeatslocated in exon 3 (Gan et al (1990) Biochemistry, 29, 9432-9440). Withinthe human population 3 length variants of the profilaggrin gene havebeen identified, encoding multimers of 10, 11 or 12 repeats. It has beenshown that both profilaggrin alleles are expressed at approximatelyequal levels, ie, that expression from the profilaggrin gene isbi-allelic (Nirunsuksiri et al (1998) Journal of InvestigativeDermatology, 110(6), 854-861).

The allelic differences of profilaggrin genes in individuals affected byichthyosis vulgaris (IV), a scaling skin disorder inherited as adominant trait, were compared to unaffected, related or age- andsex-matched normal controls (Nirunsuksiri et al (1998) Journal ofInvestigative Dermatology, 110(6), 854-861). Estimation of the size andnumber of repeats was performed utilising the EcoRV restrictions sitesthat flank the entire coding region. The number of filaggrin domains wasshown to vary between 10 and 12 in both IV and control individuals andno obvious difference in the distribution of alleles was seen betweenthe two groups (Nirunsuksiri et al (1998) Journal of InvestigativeDermatology, 110(6), 854-861) suggesting that the profilaggrin genotypeof an individual has no influence on the skin condition of thatindividual. This view is further supported by Gan et al (1990,Biochemistry, 29, 9432-9440) who note that it would appear that normalterminally differentiated human epidermis is not critically dependent onthe precise amount of functional filaggrin produced from the precursorgene.

Against this background it has been surprisingly shown a correlationbetween the number of filaggrin repeats and the predisposition to dryskin. We have shown that there is a relationship between profilaggringenotype and ability of skin to withstand surfactant challenge (ie,predisposition to detergent-induced erythema). It has also beendemonstrated that there is a direct correlation between the number offilaggrin repeats and the production of NMF. An individual's ability toproduce NMF and/or predisposition to a skin condition such as dry skin,dandruff and/or detergent-induced erythema can be determined byidentifying the profilaggrin alleles present in their genome.Individuals can be grouped during clinical trials based theirprofilaggrin genotype. Individuals can be matched with appropriatecosmetic products based on their profilaggrin genotype.

SUMMARY OF THE INVENTION

Accordingly the present invention provides a method for determining thepredisposition of an individual to a skin condition comprisingidentifying the profilaggrin alleles present in the genome of an ex vivosample taken from the individual.

The invention also provides a system for determining the predispositionof an individual to a skin condition comprising means for identifyingthe profilaggrin alleles present in the genome of a sample taken fromthe individual.

The invention also provides a method for increasing NMF productionand/or treating or preventing dry skin and/or dandruff comprisingadministering a polypeptide comprising the sequence of a profilaggrin,or a variant or fragment thereof or a polynucleotide that encodes any ofthese. In a preferred embodiment the method is for treating orpreventing detergent-induced erythema. Preferably the polypeptidecomprises the sequence of a profilaggrin allele having 12 filaggrinrepeats.

The invention also provides a polypeptide comprising the sequence of aprofilaggrin allele, or a variant or fragment thereof or apolynucleotide that encodes any of these for use in medicine. Preferablythe polypeptide comprises the sequence of a profilaggrin allele having12 filaggrin repeats.

The invention also provides the use of polypeptide comprising thesequence of a profilaggrin allele, or a variant or fragment thereof or apolynucleotide that encodes any of these in the manufacture of acomposition (for example a cosmetic composition or a medicament) fortreating a skin condition. Preferably the polypeptide comprises thesequence of a profilaggrin allele having 12 filaggrin repeats.

The invention also provides a polynucleotide comprising the SEQ ID NO:1:5′ GGA TGA AGC CTA TGA CACCAC 3′ or the SEQ ID No: 2: 5′ GA CAG GAAAAG ATA ACT TCC C 3′. The invention also provides the use of such apolynucleotide in determining the predisposition of an individual to askin condition.

The invention also provides a method for identifying personal careproducts that are suitable for an individual to use comprisingidentifying the profilaggrin genotype of an individual and selectingpersonal care products known to be suitable for use with the thusdetermined profilaggrin genotype.

The invention also provides a diagnostic kit comprising means foridentifying the profilaggrin alleles present in the genome of a sampletaken from the individual.

DETAILED DESCRIPTION OF THE INVENTION

The present invention addresses the need to determine how anindividual's skin will respond to conditions such as environmentalconditions, or contact with personal care products such as skin careproducts, cosmetics, cleansing products or hair care products orhousehold products such as fabric detergents, fabric softeners,dishwashing detergents and the like. Example of skin care productsinclude but are not limited to moisturisers, fake tanning preparations,sun tan lotions, massage oils, bath oils, perfumes, balms, creams, facepacks, shaving foams and gels. Examples of cosmetics include but are notlimited to lipsticks, foundation, eye-shadow, eyeliner, blusher andconcealer. Examples of cleansing products include but are not limited toshampoos (in particular antidandruff shampoos), soap, personal washproducts including shower gel and bubble bath and fabric detergents anddishwashing detergents. Examples of hair care products include but arenot limited to hair styling mousses, hair styling sprays, hair stylinggels, hair conditioners or hair colourants.

By assessing the profilaggrin genotype of an individual it is possibleto determine the individual's predisposition to a skin condition. By“profilaggrin genotype” is meant the identity of profilaggrin alleles inthe genome of the individual. Individuals tested by a method of theinvention are typically mammalian. In one embodiment the mammal may be arodent. In another embodiment the mammal may be a human. Thusindividuals tested by a method of the invention are diploid and socomprise two copies of the profilaggrin gene within their genome. If anindividual has two identical copies of a profilaggrin gene then they arehomozygous for that allele. If an individual has two different copies ofa profilaggrin gene, i.e. one is polymorphic to the other, then theindividual is heterozygous for that allele. By “predisposition” is meantthat the presence of an individual profilaggrin allele in the genome ofan individual, or the combination of profilaggrin alleles present in thegenome of an individual, are associated with, or are predictive of, askin condition.

The term “skin condition” as used herein includes within its meaning allphysical parameters of the skin, including the scalp, such as moistureretention, substance production or barrier formation. In one embodimentthe term “skin condition” refers to the ability of the skin to maintainhealthy levels of NMF production. Accordingly, the invention provides amethod of determining the presdisposition of an individual to maintain ahealthy level of NMF production. To put it another way the inventionprovides a method of determining the individual's susceptibility toconditions related to aberrant NMF production. Typically skin conditionscaused or exacerbated by aberrant NMF production are caused by theproduction of less NMF than by healthy skin. Conditions associated withaberrant filaggrin and NMF production include Ichthyosis Vulgaris. Inanother embodiment the term “skin condition” refers to dry skin. Dryskin conditions include senile/post-menopausal xerosis, surfactantinduced xerosis, winter xerosis, sunburn. In another embodiment the term“skin condition” refers to conditions of the scalp such as dandruff. Inanother embodiment the term “skin condition” refers to erythema, such asdetergent-induced erythema.

Thus the method of the invention provides a means for categorisingindividuals by the characteristics of their skin. This can be useful inboth therapeutic and non-therapeutic applications. In one embodimentmethods of the invention are used for therapeutic applications. Inanother embodiment methods of the invention are used for non-therapeuticapplications, such as cosmetic applications.

Therapeutic applications of methods of the invention include means ofdiagnosing the cause of a medical skin condition. Accordingly the methodof treatment for the medical skin condition can be tailored tocomplement the individual's phenotype. Therapeutic applications ofmethods of the invention also include means of determining whether anindividual's skin is likely to react adversely to a pharmaceuticalpreparation, such as a topically administered pharmaceuticalpreparation. In that case the individual can be matched to a particularpharmaceutical preparation in order to provide maximum therapeuticbenefit whilst minimising or avoiding any undesirable effects on thecondition of the individual's skin.

Non-therapeutic applications of methods of the invention include meansof grouping individuals for the purposes of trials for agents, forexample, cosmetics or any other form of preparation introduced to thebody. This can be useful for interpreting the results obtained from suchtrials, for example where the reaction of the skin of differentindividuals during the trial is not uniform. The heterogeneity ofresponses might be interpreted more clearly by grouping or stratifyingindividuals according to their predisposition to skin conditions. Theskilled person will appreciate that using this method it may be possibleto develop agents that are suitable for use with some individuals butnot suitable with others. Accordingly a panel of agents can be built up,which panel includes different agents having suitability for use withdifferent individuals. Following the trials, individuals wishing to usesuch an agent can use a method of the invention to determine whichagents are most suitable for use based on their own predisposition toskin conditions. Thus the method of the invention allows an individualto be matched with a personal care product such as those listed above.

Methods of identifying the profilaggrin genotype of an individual areperformed on biological material of the individual. Preferably thebiological material is removed from the individual prior to performingthe method of identification. In other words, typically the biologicalmaterial is ex vivo. The ex vivo material may be further cultured invitro prior to performing the method.

An ex vivo sample may comprise tissue or cells taken from any part ofthe body. A preferred ex vivo sample comprises material taken from thecirculatory system, or material taken from a bodily cavity, such as theoral cavity. A particularly preferred ex vivo sample is a saliva sample.The alleles present in an individual can be determined from a salivasample using methods known in the art, such as that described in Schieand Wilson (1997, Journal of Immunological Methods, 208, 91-101).

Accordingly the ex vivo sample may be provided by an individual withoutneed for specialised collection means. For example, a saliva sample orbuccal swab can be simply provided by the individual prior to testing.

The profilaggrin gene and protein are well known in the art and aredescribed above and in Gan et al (1990, Biochemistry, 29, 9432-9440).Numerous profilaggrin sequences have been deposited in publiclyaccessible databases.

A profilaggrin gene comprises multiple filaggrin repeats, usually 10, 11or 12 repeats. The filaggrin repeats are typically of the same length(972 bp, 324 amino acids in humans) as each other, although this is lesstypical of filaggrin repeats at the 5′- and 3′-ends of the mRNA. Thefilaggrin repeats may display considerable sequence variation, typicallyof from 0-50%, more typically of from 2-30%, yet more typically of from10-15%, between repeats on the same allele and between differentalleles. Usually variations are attributable to a single-base change butmay also involve a change in charge (Gan et al (1990) Biochemistry, 29,9432-9440). A consensus amino acid sequence map of a human filaggrinrepeat is known (Gan et al (1990) Biochemistry, 29, 9432-9440) andpreferably a filaggrin repeat will have at least 50%, more preferably atleast 75%, more preferably 90%, yet more preferably at least 95%sequence identity to that consensus sequence or a variant of theconsensus sequence shown in Gan et al (1990, Biochemistry, 29,9432-9440). Normally the amino acid sequences encoding the amino andcarboxy termini are more conserved, as are the 5′ and 3′ DNA sequencesflanking the coding portions of the gene (Presland et al (1992) J BiolChem, 267(33), 23772-23781).

The presence of different profilaggrin alleles in the genome of anindividual can be identified by methods well known in the art fordistinguishing between macromolecules with divergent structures. Theterm “allele” as used herein with respect to profilaggrin refers to anyprofilaggrin gene comprising a polymorphism. In a preferred embodimentthe term “allele” with respect to profilaggrin refers to a profilaggringene identifiable by the number of filaggrin repeats it encodes.However, the skilled person will appreciate that many otherpolymorphisms of the profilaggrin gene are possible and all profilaggrinalleles are included within the scope of the invention. For example, thedifferent phenotypes observed between individuals having profilaggrinalleles encoding profilaggrin with 10, 11 or 12 filaggrin repeats may bea direct result of the differences in production of filaggrin. However,the skilled person will appreciate that the number of filagrgin repeatsmay instead be a ‘marker’ for some other sequence polymorphism in thedifferent profilaggrin alleles, or in another gene within the epidermaldifferentiation complex. Thus the phenotype may not be directly relatedto the number of filaggrin repeats present. Thus it will be appreciatedthat methods described herein will be suitable to identify differencesbetween any profilaggrin alleles and that the invention is notrestricted to polymorphism in respect of the number of filaggrinrepeats.

Accession number M60494 identifies the 3′ end of a human profilaggringene (and is the amino terminus sequence of profilaggrin disclosed inGan et al (1990, Biochemistry, 29, 9432-9440) and includes bothEF-hands, intron 2, the N-terminus and truncated repeat and the firstfull filaggrin repeat ending at linker 2.

Accession number L01089 identifies exons 2-3 of the human profilaggringene (and is the amino terminus sequence of profilaggrin disclosed inPresland et al (1992) J Biol Chem, 267(33), 23772-23781). It containsEF-hand 2 (EF-hand 1 is in exon 1 L01088), the truncated repeat, thefirst linker and about half of the first full filaggrin repeat.

Accession number AH003056 identifies the carboxy terminus sequence ofprofilaggrin (it combines M60501.1, M60502.1 and M60503.1 as publishedby Gan et al (1990, Biochemistry, 29, 9432-9440) and includes the lastfull filaggrin repeat, truncated repeat, c-terminus and poly A tail.

Typically an allele may be identified at the polynucleotide level, suchas by analysis of genomic DNA or mRNA. The skilled person is well awareof methods for determining the presence or absence of differentpolynucleotides. Methods known for determining the presence or absenceof particular RNA sequences include northern blots, reversetranscription and PCR (RT-PCR) and ribonuclease protection assays(Sambrook and Russell, (2001) Molecular Cloning: A Laboratory Manual.3rd edition, Cold Spring Harbour Laboratory Press, New York, USA).Methods known for determining the presence or absence of particular DNAsequences include sequencing, Southern blots, PCR amplification ofgenomic DNA and analysis of restriction fragment length polymorphisms(RFLPs). See Sambrook and Russell (2001, Molecular Cloning: A LaboratoryManual. 3rd edition, Cold Spring Harbour Laboratory Press, New York,USA), Innis et al, (1995, PCR Strategies, Academic Press, Inc.: NY);Dieffenbach et al (1995, PCR Primer: A Laboratory Manual, New York: ColdSpring Harbor Press). DNA sequence analysis may also be achieved bydetecting alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Differences can also bevisualized by high resolution gel electrophoresis or distinguishedaccording to differences in DNA sequence melting points. See, e.g.,Myers et al (1982, Science, 230, 1242). Methods for detecting thepresence of specific sequences include detection techniques such asfluorescence-based detection methods, immune-based assays such as RIA,antibody staining such as Western blot analysis or in situhybridization, using appropriately labeled probe.

Sequences useful for constructing probes suitable for use in detectingthe presence of a sequence of interest include any nucleic acid sequencehaving at least about 50%, preferably at least 70%, more preferably atleast 80% or greater sequence identity or homology with the sequence ofa known profilaggrin gene or fragment thereof by a Blast search.“Percent (%) sequence identity” or “percent (%) sequence homology” isdefined as the percentage of nucleic acid residues in a candidatesequence that are identical with the nucleic acid residues of thesequence of interest, after aligning the sequences and introducing gaps,if necessary to achieve maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Methods for performing sequence alignment and determiningsequence identity are known in the art, may be performed without undueexperimentation, and calculations of % identity values may be obtainedfor example, using available computer programs such as WU-BLAST-2(Altschul et al, 1996, Methods in Enzymology 266, 460-480). One mayoptionally perform the alignment using set default parameters in thecomputer software program (Blast search, MacVector and Vector NTI).Based upon the restriction map of a particular allele, a banding patterncan be predicted when the Southern blot is hybridized with a probe whichrecognizes the sequence of interest. The level of stringency ofhybridization used can vary depending upon the level of sensitivitydesired, a particular probe characteristic, such as probe length and/orannealing temperature, or degree of homology between probe sequence andsequence of interest. Therefore, considerations of sensitivity andspecificity will determine stringency of hybridization required for aparticular assay.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperatures. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. For additional details and explanation ofstringency of hybridization reactions, see Ausubel et al (1995, CurrentProtocols in Molecular Biology, Wiley Interscience Publishers) orProtocols Online (URL: www.protocol-online.net/molbio/index.htm).

“Stringent conditions” or “high-stringency”, as defined herein, may beidentified by those that: (1) use low ionic strength and hightemperature for washing, for example 0.1×SSC, 0.2% SDS at 65-70° C.

“Moderately-stringent conditions” may be identified as described bySambrook and Russell (2001, Molecular Cloning: A Laboratory Manual, 3rdedition), and include the use of washing solution and hybridisationconditions (e.g. temperature, ionic strength, and % SDS) less stringentthat those described above. An example of moderately stringentconditions is 0.2×SSC, 0.1% SDS at 58-65° C. The skilled artisan willrecognise how to adjust temperature, ionic strength, etc. as necessaryto accommodate factors such as probe length, degree of homology betweenprobe and target site and the like. Therefore, in addition to thesequence of interest, it is contemplated that additional or alternativeprobe sequences which vary from that of the sequence of interest willalso be useful in screening for the sequence of interest.

In a preferred embodiment profilaggrin alleles are identified by thenumber of filaggrin repeats present. Thus typically the method ofidentifying the profilaggrin alleles present in the genome of anindividual comprises determining whether the alleles present have 10, 11or 12 filaggrin repeats.

In one preferred embodiment allele identification is performed usingPCR. Forward and reverse primers are prepared using techniques wellknown in the art and comprise a sequence based on an upstream region anda downstream region, respectively, relative to the sequence of theprofilaggrin gene coding sequence encoding polymorphic filaggrinrepeats. Preferably the upstream and downstream regions chosen fordesign of primers will be substantially conserved between differentalleles. “Substantially conserved” includes within its meaning sequenceshaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequenceidentity. Thus primers can be designed for binding to similar butnon-identical sequences, for example by using degenerate primers or byincluding nucleotides that have a reduced specificity for the purposesof complementarity, such as inosine, within the primer. Preferably one,or more preferably both, of the forward and reverse primers are 100%identical to the upstream and/or downstream regions of each profilaggrinallele.

Upstream and downstream primers can be derived from the sequencespresent in publicly available databases. For example, one of the primersused in the examples below is designed in accordance with bases3112-3132 of the sequence identified by accession number L01089(equivalent to bases 1530-1551 of the sequence identified by accessionnumber M60494), whereas the other primer is designed in accordance withbases 3341-3361) of the sequence identified by accession numberAH003056.

Amplification of profilaggrin alleles using these primers will producedifferent sized products dependent of the number of encoded filaggrinrepeats. It is thought that a 10 repeat allele should yield a fragmentof 11,610 bp, an 11 repeat allele should yield a fragment of 12,582 bpand a 12 repeat allele should yield a fragment of 13,554 bp. Thus apreferred upstream region used for design of the forward primer is atleast a part of the region of the profilaggrin gene encoding the aminoterminus or the 5′ DNA sequence upstream of the coding portions of thegene. Preferably the sequence of the forward primer is SEQ ID NO: 1:5′GGA TGA AGC CTA TGA CACCAC 3′.

A preferred downstream region used for design of the reverse primer isat least a part of the region of the profilaggrin gene encoding thecaroxy terminus or the 3′ DNA sequence downstream of the coding portionsof the gene. Preferably the sequence of the reverse primer is SEQ ID NO:2: 5′ GA CAG GAA AAG ATA ACT TCC C 3′.

The PCR reaction is performed in order to amplify DNA obtained from thebiological material from the sample taken from the individual. In oneembodiment the DNA is genomic DNA extracted from the biologicalmaterial. In another embodiment the DNA is cDNA which has been reversetranscribed from RNA, typically mRNA, which RNA has been extracted fromthe biological material. Methods for extracting genomic DNA, methods forextracting RNA, methods for extracting mRNA and methods for reversetranscription of RNA are well known in the art, for example see Sambrookand Russell (2001, Molecular Cloning: A Laboratory Manual. 3rd edition,Cold Spring Harbour Laboratory Press, New York, USA).

In a preferred embodiment the DNA is genomic DNA and the sample is asaliva sample or buccal swab. Methods for extracting DNA from salivasamples and buccal swabs are known in the art (Schie and Wilson (1997)Journal of Immunological Methods, 208, 91-101).

The PCR reaction can be performed under conditions well known in the artor as suggested by the manufacturer of a commercially available PCR kit.For example, amplification may be performed using from 0.1 to 30 μg/mlDNA substrate. Amplification may be performed using from 2 μM to 2 mMdNTPs. Amplification may be performed using from 2 μM to 2 mM forwardand reverse primers. Amplification may be performed using and from 17 μMto 170 mM Mg²⁺. In a preferred embodiment amplification is performedusing about 200 μM dNTPs. In a preferred embodiment amplification isperformed using about 200 μM forward and reverse primers. In a preferredembodiment amplification is performed using about 1.7 mM Mg²⁺. By“about” is meant that the concentration used varies by no more than 50%,25%, 10% or 5% from the concentration stated. Most preferably the PCRreaction is performed essentially as described in the exemplifiedmethods below.

PCR products can then be analysed by any suitable method. Typically thePCR products are analysed by size fractionation, usually using gelelectrophoresis performed in accordance with techniques well known inthe art (see Sambrook and Russell (2001) Molecular Cloning: A LaboratoryManual. 3rd edition, Cold Spring Harbour Laboratory Press, New York,USA). Most preferably the PCR products are analysed essentially asdescribed in the exemplified methods below.

Other methods suitable for identifying the profilaggrin alleles presentin the genome of an individual include allele specific hybridisation;allele specific oligonucleotide hybridisation; and primer specificextension.

Allele specific hybridization uses probes overlapping a region of atleast one profilaggrin allele and having about 5, 10, 20, 25 or 30nucleotides around a polymorphic region. In a preferred embodiment,several probes capable of hybridizing specifically to other profilagginalleles are attached to a solid phase support, e.g. a “chip” (which canhold up to about 250,000 oligonucleotides).

Oligonucleotides can be bound to a solid support by a variety ofprocesses, including lithography. Mutation detection analysis usingthese chips comprising oligonucleotides, also terms “DNA probe arrays”is described e.g., in Cronin et al (1996, Human Mutation 7, 244). In oneembodiment, a chip comprises all the allelic variants of at least onepolymorphic region of a profilaggrin gene. The solid phase support isthen contacted with a test nucleic acid and hybridization to thespecific probes is detected. Accordingly, the identity of numerousallelic variants of one or more genes can be identified in a simplehybridization experiment.

These techniques may also comprise the step of amplifying the nucleicacid before analysis. Amplification techniques are known to those ofskill in the art and include, but are not limited to cloning, polymerasechain reaction (PCR), polymerase chain reaction of specific alleles(ASA), ligase chain region (LCR), nested polymerase chain reaction, selfsustained sequence replication (Guatelli et al (1990) Proc Natl Acad SciUSA 87, 1874-1878), transcriptional amplification system (Kwoh et al(1989) Proc Natl Acad Sci USA 86, 1173-1177), and Q-Beta Replicase(Lizardi (1988) Bio/Technology 6, 1197).

Amplification products may be assayed in a variety of ways, includingsize analysis, restriction digestion followed by size analysis,detecting specific tagged oligonucleotide primers in the reactionproducts, allele-specific oligonucleotide (ASO) hybridization, allelespecific 5′ exonuclease detection, sequencing, hybridization, and thelike.

In a merely illustrative embodiment a method of identifying profilaggrinalleles includes the steps of (i) isolating nucleic acid (e.g., genomic,RNA or both) from the cells of a sample collected from an individual(ii) contacting the nucleic acid sample with one or more primers whichspecifically hybridize 5′ and 3′ to at least one polymorphism in theprofilaggrin allele under conditions such that hybridization andamplification of the polymorphic region of the allele occurs, and (iii)detecting the amplification product. These detection schemes areespecially useful for the detection of nucleic acid molecules if suchmolecules are present in very low numbers.

An allele of profilaggrin may be identified by alterations inrestriction enzyme cleavage patterns. For example, sample and controlDNA is isolated, amplified (optionally) digested with one or morerestriction endonucleases, and fragment length sizes are determined, forexample by gel electrophoresis.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the allele. Exemplarysequencing reactions include those based on techniques developed byMaxim and Gilbert (1997, Proc Natl Acad Sci USA 74, 560) or Sanger et al(1977, Proc Nat Acad Sci USA 74, 5463). It is also contemplated that anyof a variety of automated sequencing procedures may be utilized whenperforming the subject assays (see, for example Biotechniques (1995) 19,448), including sequencing by mass spectrometry (e.g. WO 94/16101; Cohenet al (1996) Adv Chromatogr 36, 127-162; and Griffin et al (1993) ApplBiochem Biotechnol 38, 147-159). It will be evident to one of skill inthe art that, for certain embodiments, the occurrence of only one, twoor three of the nucleic acid bases need be determined in the sequencingreaction. For instance, A-track or the like, e.g., where only onenucleic acid is detected, can be carried out.

A profilaggrin allele may be identified by using cleavage agents (suchas nuclease, hydroxylamine or osmium tetroxide and with piperidine) todetect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNA heteroduplexes(Myers et al (1985) Science 230, 1242). In general, the art technique of“mismatch cleavage” starts by providing heteroduplexes formed byhybridizing (labeled) RNA or DNA containing the wild-type allele with asample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex such as which will existdue to base pair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digest the mismatched regions.In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size, for example usingdenaturing polyacrylamide gel to determine the site of mutation. See,for example, Cotton et al (1988) Proc Natl Acad Sci USA 85, 4397; andSaleeba et al (1992) Methods Enzymol 217, 286-295. In a preferredembodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes). For example, the mutYenzyme of E. coli cleaves A at G/A mismatches and the thymidine DNAglycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.(1994) Carcinogenesis 15, 1657-1662). According to an exemplaryembodiment, a probe based on a chosen profilaggrin allele is hybridizedto a cDNA or other DNA product from a test cell(s). The duplex istreated with a DNA mismatch repair enzyme, and the cleavage products, ifany, can be detected from electrophoresis protocols or the like. See,for example, U.S. Pat. No. 5,459,039.

Examples of other techniques for detecting alleles include, but are notlimited to, selective oligonucleotide hybridization, or selective primerextension. For example, oligonucleotide primers may be prepared in whichthe known mutation or nucleotide difference (e.g., in allelic variants)is placed centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki et al(1986) Nature 324, 163; Saiki et al (1989) Proc Natl Acad Sci USA 86,6230). Such allele specific oligonucleotide hybridization techniques maybe used to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hyrbidized with labeledtarget DNA.

In another embodiment, identification of a profilaggrin allele may becarried out using an oligonucleotide ligation assay (OLA), as described,e.g., in U.S. Pat. No. 4,998,617 and in Landegren et al (1988, Science241, 1077-1080). The OLA protocol uses two oligonucleotides which aredesigned to be capable of hybridizing to abutting sequences of a singlestrand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson et al have described a nucleic acid detection assay thatcombines attributes of PCR and OLA (Nickerson et al (1990) Proc NatlAcad Sci USA 87, 8923-27). In this method, PCR is used to achieve theexponential amplification of target DNA, which is then detected usingOLA.

Several techniques based on this OLA method have been developed and canbe used to detect profilaggrin alleles. For example, U.S. Pat. No.5,593,826 discloses an OLA using an oligonucleotide having 3′-aminogroup and a 5′-phosphorylated oligonucleotide to form a conjugate havinga phosphoramidate linkage. In another variation of OLA described in Tobe et al (1997, Nucleic Acids Res 24, 3728), OLA combined with PCRpermits typing of two alleles in a single microtiter well. By markingeach of the allele-specific primers with a unique hapten, i.e.digoxigenin and fluorescein, each OLA reaction can be detected by usinghapten specific antibodies that are labeled with different enzymereporters, alkaline phosphatase or horseradish peroxidase. This systempermits the detection of the two alleles using a high throughput formatthat leads to the production of two different colours.

Once the profilaggrin genotype of an individual has been determined,that individual can be categorised as having a high or lowpredisposition to a skin condition. Thus methods of the invention can beused to identify the profilaggrin genotype of an individual in order todetermine that individual's predisposition to a skin condition.Accordingly the invention provides a system for determining thepredisposition of an individual to a skin condition comprising means foridentifying the profilaggrin alleles present in the genome of a sampletaken from the individual.

In one embodiment the invention provides a polynucleotide comprising thesequence of a primer for use in amplifying the region of theprofilaggrin gene comprising a polymorphism. In a preferred embodimentthe primer is SEQ ID NO: 1: 5′ GGA TGA AGC CTA TGA CACCAC 3′ or SEQ IDNO: 2: 5′ GA CAG GAA AAG ATA ACT TCC C 3′.

The invention also provides for the use of a primer of the invention ina method of determining the predisposition of an individual to a skincondition as described above. Thus kits and assay components comprisingPCR primers and oligonucleotides for hybridisation as described aboveform further aspects of the invention.

The primer kit of the present invention is useful for identifyingprofilaggrin alleles using the polymerase chain reaction. The kitcomprises a set of pairs of single stranded DNA primers which can beannealed to sequences flanking the polymorphism and within orsurrounding the profilaggrin gene on the relevant chromosome in order toprime amplifying DNA synthesis of the gene itself. The complete set mayallow synthesis of all of the nucleotides of the profilaggrin allelecoding sequences, ie the exons, or may allow synthesis of less than theentire coding region. The set of primers preferably allows synthesis ofboth intron and exon sequences, as allelic variations may be found in aprofilaggrin gene intron. The kit can also contain DNA polymerase,preferably a thermophilic DNA polymerase, more preferably Taqpolymerase, yet more preferably Elongase (GIBCOBRL Life Technologies)and suitable reaction buffers. Such components are known in the art.

In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme sites appended to their 5′ ends.Thus, all nucleotides of the primers are derived from profilaggrin genesequences or sequences adjacent to that gene except the few nucleotidesnecessary to form a restriction enzyme site. Such enzymes and sites arewell known in the art. The primers themselves can be synthesized usingtechniques which are well known in the art. Generally, the primers canbe made using synthesizing machines which are commercially available.Given the sequences of the profilaggrin allelic variations which areknown in the art, design of particular primers is well within the skillof the art.

Kits of the invention optionally further comprise a personal careproduct, such as cosmetic preparation as described above, whichpreparation is suitable for use on an individual having a particularprofilaggrin genotype.

An example of a kit according to the invention may include a means ofnucleic acid isolation, such as QIAamp DNA Blood Midi Kit or EpicentreBuccalAmp kit and associated equipment such as a centrifuge, means forDNA quantitation such as a spectrophotometer, means for performing a PCRreaction such as a thermal cycler and means for analysing PCR productssuch as a gel electrophoresis kit. However, many or all of these itemswill be readily available in a molecular biology laboratory. Therefore,a kit according to the invention may comprise dNTPs suitable fordilution to a 200 μM final concentration, a pair of oligonucleotideprimers such as SEQ ID NO: 1: 5′ GGA TGA AGC CTA TGA CACCAC 3′ and SEQID NO: 2: 5′ GA CAG GAA AAG ATA ACT TCC C 3′ magnesium chloride solutionsuitable for filution to 1.7 mM final concentration Mg²⁺ and athermostable DNA polymerase such as Elongase (GIBCOBRL LifeTechnologies). The kit may further comprise instructions for using thecomponents and may therefore include instructions for performance of thePCR cycle as follows: —

Heat reactions to 94° C. for 5 minsAdd Elongase reaction mix to 1/50 dilution (HOT START)Start programme:

 5 minutes 94° C.  1 cycle 30 seconds 94° C. 30 seconds 57° C. {closeoversize brace} 35 cycles 12 minutes 68° C. soak  4° C.

A kit of the invention may further comprise polynucleotides having knownsizes for comparison with and sizing of the PCR products. Suchpolynucleotides may be, for example, extension ladder markers (such asproduced by GIBCO-BRL) or a reference genomic DNA containg previouslyidentified variants.

The present invention also contemplates a method for cosmetic treatmentcomprising determining the profilaggrin genotype of an individual by amethod as described above in order to identify a cosmetic preparationsuitable for use with an individual having that profilaggrin genotypeand using the thus identified cosmetic preparation on that individual.The use of the cosmetic preparation involves use in the normal manner.Typically this will involve topical application to the skin or scalp ofthe individual. The cosmetic preparation may be any preparation asdescribed above.

The present invention also contemplates a method for treating a skincondition by influencing the type and/or availability of profilaggrinalleles in the epidermis of an individual. This may be achieved byadministering a polypeptide comprising the sequence of a profilaggrinalleles or a variant or fragment thereof. This may also be achieved byadministering polynucleotide comprising a sequence that encodes aprofilaggrin allele or a variant or fragment thereof. Thus the presentinvention contemplates a method of modifying, preferably increasing, NMFproduction in the epidermis of an individual. The present invention alsocontemplates a method of treating dry skin and/or detergent-inducederythema. The present invention also contemplates a method of treatingdandruff. The polypeptide or polynucleotide may be formulated with acosmetic preparation to enhance the beneficial effects of that cosmeticor to ameliorate the undesirable effects of that cosmetic. Thus in apreferred embodiment, a method of the invention can increaseprofilaggrin production. Preferably the profilaggrin allele that isproduced has 12 filaggrin repeats.

“Fragments” and “variants” include within their meaning polypeptidesthat are useful to prepare antibodies which will specifically bind aprofilaggrin or mutant forms thereof. It is well known that sequencedivergence occurs in the filaggrin repeats (Gan et al (1990)Biochemistry, 29, 9432-9440).

A profilaggrin “variant” includes within its meaning a polypeptidewherein at one or more positions there have been amino acid insertions,deletions, or substitutions, either conservative or non-conservative,provided that such changes result in a protein whose basic properties,for example binding activity (type of and affinity), thermostability,activity in a certain pH-range (pH-stability) have not significantlybeen changed. “Significantly” in this context means that one skilled inthe art would say that the properties of the variant may still bedifferent but would not be unobvious over the ones of the originalprotein. By “conservative substitutions” is intended combinations suchas Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; andPhe, Tyr.

A “fragment” is less than 100% of the whole polypeptide. For example, atleast 99%, 98%, 95%, 90%, 80%, 60%, 40%, 30%, 25% or 20% of the wholeprofilaggrin protein.

It will be recognised by those skilled in the art that the polypeptideof the invention may be modified by known polypeptide modificationtechniques. These include the techniques disclosed in U.S. Pat. No.4,302,386 issued 24 Nov. 1981 to Stevens, incorporated herein byreference. Such modifications may alter, preferably enhance theimmunogenicity of the antigen, or they may have no effect on suchimmunogenicity. For example, a few amino acid residues may be changed.

Alternatively, smaller polypeptides corresponding to antigenic parts ofthe polypeptide may be chemically synthesised by methods well known inthe art. These include the methods disclosed in U.S. Pat. No. 4,290,944issued 22 Sep. 1981 to Goldberg, incorporated herein by reference.

Thus, the polypeptide for use in methods of the invention includes aclass of modified polypeptides, including synthetically derivedpolypeptides or fragments of the original polypeptide, having commonelements of origin, structure, and immunogenicity that are within thescope of the present invention.

An isolated polynucleotide for use in a method of the invention maycomprise a sequence that encodes a profilaggrin gene or a variant orfragment thereof as described above for use in a method of theinvention. As used herein, the term “isolated” means that the gene is inisolation from at least most of the human chromosome on which it isfound, in other words the gene is not claimed in the form in which ithas previously existed. Thus, the gene of the invention includes thegene when that gene has been cloned into a bacterial vector, such as aplasmid, or into a viral vector, such as a bacteriophage, provided thatsuch clones are in isolation from clones constituting a DNA library ofthe relevant chromosome.

The “gene” may comprise the promoter and/or other expression-regulatingsequences which normally govern its expression and it may compriseintrons, or it may consist of the coding sequence only, for example acDNA sequence.

Alternatively antisense polynucleotides may be used in a method of theinvention. Antisense polynucleotides are single-stranded nucleic acids,which can specifically bind to a complementary nucleic acid sequence. Bybinding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, orRNA-DNA duplex is formed. These nucleic acids are often termed“antisense” because they are complementary to the sense or coding strandof the gene. Recently, formation of a triple helix has proven possiblewhere the polynucleotide is bound to a DNA duplex. It was found thatpolynucleotides could recognise sequences in the major groove of the DNAdouble helix. A triple helix was formed thereby. This suggests that itis possible to synthesise sequence-specific molecules which specificallybind double-stranded DNA via recognition of major groove hydrogenbinding sites.

By binding to a profilaggrin target nucleic acid, the abovepolynucleotides can inhibit the function of the target nucleic acid.This could, for example, be a result of blocking the transcription,processing, poly(A)addition, replication, translation, or promotinginhibitory mechanisms of the cells, such as promoting RNA degradations.

Antisense polynucleotides may be prepared in the laboratory and thenintroduced into cells, for example by microinjection or uptake from thecell culture medium into the cells, or they are expressed in cells aftertransfection with plasmids or retroviruses or other vectors carrying anantisense gene. Antisense polynucleotides were first discovered toinhibit viral replication or expression in cell culture for Rous sarcomavirus, vesicular stomatitis virus, herpes simplex virus type 1, simianvirus and influenza virus. Since then, inhibition of mRNA translation byantisense polynucleotides has been studied extensively in cell-freesystems including rabbit reticulocyte lysates and wheat germ extracts.Inhibition of viral function by antisense polynucleotides has beendemonstrated in vitro using polynucleotides which were complementary tothe AIDS HIV retrovirus RNA (Goodchild, J. 1988 “Inhibition of HumanImmunodeficiency Virus Replication by Antisense Oligodeoxynucleotides”,Proc. Natl. Acad. Sci. (USA) 85(15), 5507-11). The Goodchild studyshowed that polynucleotides that were most effective were complementaryto the poly(A) signal; also effective were those targeted at the 5N endof the RNA, particularly the cap and 5N untranslated region, next to theprimer binding site and at the primer binding site. The cap, 5Nuntranslated region, and poly(A) signal lie within the sequence repeatedat the ends of retrovirus RNA (R region) and the polynucleotidecomplementary to these may bind twice to the RNA.

Typically, antisense polynucleotides are 15 to 35 bases in length. Forexample, 20-mer polynucleotides have been shown to inhibit theexpression of the epidermal growth factor receptor mRNA (Witters et al,Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer polynucleotides havebeen shown to decrease the expression of adrenocorticotropic hormone bygreater than 90% (Frankel et al, J Neurosurg 91:261-7 (1999)). However,it is appreciated that it may be desirable to use polynucleotides withlengths outside this range, for example 10, 11, 12, 13, or 14 bases, or36, 37, 38, 39 or 40 bases.

The aforementioned polypeptides and polynucleotides or a formulationthereof may be administered by any conventional method includingtopically to the site of the skin condition, orally or by parenteral (egsubcutaneous or intramuscular) injection. The treatment may consist of asingle dose or a plurality of doses over a period of time.

Polynucleotides may be administered systemically.

Alternatively the inherent binding specificity of polynucleotidescharacteristic of base pairing is enhanced by limiting the availabilityof the polynucleotide to its intended locus in vivo, permitting lowerdosages to be used and minimising systemic effects. Thus,polynucleotides may be applied locally to achieve the desired effect.The concentration of the polynucleotides at the desired locus is muchhigher than if the polynucleotides were administered systemically, andthe therapeutic effect can be achieved using a significantly lower totalamount. The local high concentration of polynucleotides enhancespenetration of the targeted cells and effectively blocks translation ofthe target nucleic acid sequences.

The polynucleotides can be delivered to the locus by any meansappropriate for localised administration of a drug. For example, asolution of the polynucleotides can be injected directly to the site orcan be delivered by infusion using an infusion pump. The polynucleotidesalso can be incorporated into an implantable device which when placedadjacent to the desired site, to permit the polynucleotides to bereleased into the surrounding locus.

The polynucleotides may be administered via a hydrogel material. Thehydrogel is non-inflammatory and biodegradable. Many such materials noware known, including those made from natural and synthetic polymers. Ina preferred embodiment, the method exploits a hydrogel which is liquidbelow body temperature but gels to form a shape-retaining semisolidhydrogel at or near body temperature. Preferred hydrogel are polymers ofethylene oxide-propylene oxide repeating units. The properties of thepolymer are dependent on the molecular weight of the polymer and therelative percentage of polyethylene oxide and polypropylene oxide in thepolymer. Preferred hydrogels contain from about 10% to about 80% byweight ethylene oxide and from about 20% to about 90% by weightpropylene oxide. A particularly preferred hydrogel contains about 70%polyethylene oxide and 30% polypropylene oxide. Hydrogels which can beused are available, for example, from BASF Corp., Parsippany, N.J.,under the tradename Pluronic®.

In this embodiment, the hydrogel is cooled to a liquid state and theoligonucleotides are admixed into the liquid to a concentration of about1 mg polynucleotides per gram of hydrogel. The resulting mixture then isapplied onto the surface to be treated, for example by spraying orpainting during surgery or using a catheter or endoscopic procedures. Asthe polymer warms, it solidifies to form a gel, and the polynucleotidesdiffuse out of the gel into the surrounding cells over a period of timedefined by the exact composition of the gel.

The polynucleotides can be administered by means of other implants thatare commercially available or described in the scientific literature,including liposomes, microcapsules and implantable devices. For example,implants made of biodegradable materials such as polyanhydrides,polyorthoesters, polylactic acid and polyglycolic acid and copolymersthereof, collagen, and protein polymers, or non-biodegradable materialssuch as ethylenevinyl acetate (EVAc), polyvinyl acetate, ethylene vinylalcohol, and derivatives thereof can be used to locally deliver thepolynucleotides. The polynucleotides can be incorporated into thematerial as it is polymerised or solidified, using melt or solventevaporation techniques, or mechanically mixed with the material. In oneembodiment, the polynucleotides are mixed into or applied onto coatingsfor implantable devices such as dextran coated silica beads, stents, orcatheters.

The dose of polynucleotides is dependent on the size of thepolynucleotides and the purpose for which is it administered. Ingeneral, the range is calculated based on the surface area of tissue tobe treated. The effective dose of polynucleotide is somewhat dependenton the length and chemical composition of the polynucleotides but isgenerally in the range of about 30 to 3000 μg per square centimetre oftissue surface area.

The polynucleotides may be administered systemically for cosmetic,therapeutic and prophylactic purposes. The polynucleotides may beadministered by any effective method, for example, parenterally (egintravenously, subcutaneously, intramuscularly) or by oral, nasal orother means which permit the oligonucleotides to access and circulate inthe patient's bloodstream. Polynucleotides administered systemicallypreferably are given in addition to locally administeredpolynucleotides, but also have utility in the absence of localadministration. A dosage in the range of from about 0.1 to about 10grams per administration to an adult human generally will be effectivefor this purpose.

It will be appreciated that antisense agents also include largermolecules which bind to said profilaggrin mRNA or genes andsubstantially prevent expression of said profilaggrin mRNA or genes andsubstantially prevent expression of said profilaggrin protein. Thus,expression of an antisense molecule which is substantially complementaryto said profilaggrin mRNA is envisaged as part of the invention.

The said larger molecules may be expressed from any suitable geneticconstruct as is described below and delivered to the patient. Typically,the genetic construct which expresses the antisense molecule comprisesat least a portion of the said profilaggrin cDNA or gene operativelylinked to a promoter which can express the antisense molecule in thecell.

Although genetic constructs for delivery of polynucleotides can be DNAor RNA it is preferred if it is DNA.

Preferably, the genetic construct is adapted for delivery to a humancell.

Means and methods of introducing a genetic construct into a cell in ananimal body are known in the art. For example, the constructs of theinvention may be introduced into cells by any convenient method, forexample methods involving retroviruses, so that the construct isinserted into the genome of the cell. For example, in Kuriyama et al(1991) Cell Struc. and Func. 16, 503-510 purified retroviruses areadministered. Retroviral DNA constructs comprising a polynucleotide asdescribed above may be made using methods well known in the art. Toproduce active retrovirus from such a construct it is usual to use anecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle'smedium (DMEM) containing 10% foetal calf serum (FCS). Transfection ofthe cell line is conveniently by calcium phosphate co-precipitation, andstable transformants are selected by addition of G418 to a finalconcentration of 1 mg/ml (assuming the retroviral construct contains aneo^(R) gene). Independent colonies are isolated and expanded and theculture supernatant removed, filtered through a 0.45 μm pore-size filterand stored at −70° C. For the introduction of the retrovirus into thetumour cells, it is convenient to inject directly retroviral supernatantto which 10 μg/ml Polybrene has been added. For tumours exceeding 10 mmin diameter it is appropriate to inject between 0.1 ml and 1 ml ofretroviral supernatant; preferably 0.5 ml.

Alternatively, as described in Culver et al (1992) Science 256,1550-1552, cells which produce retroviruses are injected. Theretrovirus-producing cells so introduced are engineered to activelyproduce retroviral vector particles so that continuous productions ofthe vector occurred within the tumour mass in situ. Thus, proliferatingepidermal cells can be successfully transduced in vivo if mixed withretroviral vector-producing cells.

Targeted retroviruses are also available for use in the invention; forexample, sequences conferring specific binding affinities may beengineered into pre-existing viral env genes (see Miller & Vile (1995)Faseb J. 9, 190-199 for a review of this and other targeted vectors forgene therapy).

Other methods involve simple delivery of the construct into the cell forexpression therein either for a limited time or, following integrationinto the genome, for a longer time. An example of the latter approachincludes liposomes (Nassander et al (1992) Cancer Res. 52, 646-653).

For the preparation of immuno-liposomes MPB-PE(N-[4-(p-maleimidophenyl)butyryl]-phosphatidylethanolamine) issynthesised according to the method of Martin & Papahadjopoulos (1982)J. Biol. Chem. 257, 286-288. MPB-PE is incorporated into the liposomalbilayers to allow a covalent coupling of the antibody, or fragmentthereof, to the liposomal surface. The liposome is conveniently loadedwith the DNA or other genetic construct of the invention for delivery tothe target cells, for example, by forming the said liposomes in asolution of the DNA or other genetic construct, followed by sequentialextrusion through polycarbonate membrane filters with 0.6 μm and 0.2 μmpore size under nitrogen pressures up to 0.8 MPa. After extrusion,entrapped DNA construct is separated from free DNA construct byultracentrifugation at 80 000×g for 45 min. Freshly preparedMPB-PE-liposomes in deoxygenated buffer are mixed with freshly preparedantibody (or fragment thereof) and the coupling reactions are carriedout in a nitrogen atmosphere at 4° C. under constant end over endrotation overnight. The immunoliposomes are separated from unconjugatedantibodies by ultracentrifugation at 80 000×g for 45 min.Immunoliposomes may be injected intraperitoneally or directly into thetumour.

Other methods of delivery include adenoviruses carrying external DNA viaan antibody-polylysine bridge (see Curiel Prog. Med. Virol. 40, 1-18)and transferrin-polycation conjugates as carriers (Wagner et al (1990)Proc. Natl. Acad. Sci. USA 87, 3410-3414). In the first of these methodsa polycation-antibody complex is formed with the DNA construct or othergenetic construct of the invention, wherein the antibody is specific foreither wild-type adenovirus or a variant adenovirus in which a newepitope has been introduced which binds the antibody. The polycationmoiety binds the DNA via electrostatic interactions with the phosphatebackbone. The adenovirus, because it contains unaltered fibre and pentonproteins, is internalised into the cell and carries into the cell withit the DNA construct of the invention. It is preferred if the polycationis polylysine.

The DNA may also be delivered by adenovirus wherein it is present withinthe adenovirus particle, for example, as described below.

In an alternative method, a high-efficiency nucleic acid delivery systemthat uses receptor-mediated endocytosis to carry DNA macromolecules intocells is employed. This is accomplished by conjugating theiron-transport protein transferrin to polycations that bind nucleicacids. Human transferrin, or the chicken homologue conalbumin, orcombinations thereof is covalently linked to the small DNA-bindingprotein protamine or to polylysines of various sizes through a disulfidelinkage. These modified transferrin molecules maintain their ability tobind their cognate receptor and to mediate efficient iron transport intothe cell. The transferrin-polycation molecules form electrophoreticallystable complexes with DNA constructs or other genetic constructs of theinvention independent of nucleic acid size (from short oligonucleotidesto DNA of 21 kilobase pairs). When complexes of transferrin-polycationand the DNA constructs or other genetic constructs of the invention aresupplied to the tumour cells, a high level of expression from theconstruct in the cells is expected.

High-efficiency receptor-mediated delivery of the DNA constructs orother genetic constructs of the invention using the endosome-disruptionactivity of defective or chemically inactivated adenovirus particlesproduced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci.USA 89, 6094-6098 may also be used. This approach appears to rely on thefact that adenoviruses are adapted to allow release of their DNA from anendosome without passage through the lysosome, and in the presence of,for example transferrin linked to the DNA construct or other geneticconstruct of the invention, the construct is taken up by the cell by thesame route as the adenovirus particle.

This approach has the advantages that there is no need to use complexretroviral constructs; there is no permanent modification of the genomeas occurs with retroviral infection; and the targeted expression systemis coupled with a targeted delivery system, thus reducing toxicity toother cell types.

It will be appreciated that “naked DNA” and DNA complexed with cationicand neutral lipids may also be useful in introducing the DNA of theinvention into cells of the individual to be treated. Non-viralapproaches to gene therapy are described in Ledley (1995) Human GeneTherapy 6, 1129-1144.

Alternative targeted delivery systems are also known such as themodified adenovirus system described in WO 94/10323 wherein, typically,the DNA is carried within the adenovirus, or adenovirus-like, particle.Michael et al (1995) Gene Therapy 2, 660-668 describes modification ofadenovirus to add a cell-selective moiety into a fibre protein. Mutantadenoviruses which replicate selectively in p53-deficient human tumourcells, such as those described in Bischoff et al (1996) Science 274,373-376 are also useful for delivering the genetic construct of theinvention to a cell. Thus, it will be appreciated that a further aspectof the invention provides a virus or virus-like particle comprising agenetic construct of the invention. Other suitable viruses or virus-likeparticles include HSV, AAV, vaccinia and parvovirus.

The genetic constructs of the invention can be prepared using methodswell known in the art.

Whilst it is possible for a polypeptides or polynucleotides to beadministered alone, it is preferable to present it as a pharmaceuticalformulation, together with one or more acceptable carriers. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe compound of the invention and not deleterious to the recipientsthereof. Typically, the carriers will be water or saline which will besterile and pyrogen free.

The invention will now be described in more detail by reference to thefollowing Figures and Examples wherein:

FIG. 1 shows the identification of profilaggrin alleles in the genome ofan individual using a PCR approach to determine the number of filaggrinrepeats.

FIG. 2 shows the filaggrin repeat number per chromosome pair versusstratum corneum NMF levels. Spearman correlation: R=0.47; p=0.036; n=20.

FIG. 3 shows the Filaggrin repeat number per chromosome pair versusstratum corneum NMF levels and including scalp condition history. %dandruff with 1 or more 12 repeat alleles=67%; % dandruff with no 12repeat alleles=17%

FIG. 4 shows erythemal score 48 hours post patching with 1% SLS versusprofilaggrin allelotype. Mann-Whitney Rank Sum Test: There is astatistically significant difference between erythemal scores 24 hourspost patch when comparing no 12 repeat allele panellists with 1 or more12 repeat allele panellists (n=43) (a) SLS patch recovery—individualswith no 12 repeat alleles; (b) SLS patch recovery—individuals with 1 ormore 12 repeat alleles.

FIG. 5 shows the proportion of panellists with or without the 12 repeatallele who frequently suffer from self-perceived dry skin. Fisher'sExact test: There is a statistically significant relationship (p=0.0237)between the presence or absence of the 12 repeat allele and frequency ofdry skin (n=89).

FIG. 6 shows the proportion of panellists with or without the 11 repeatallele who displayed visual leg skin dryness. Fisher's Exact test: Thereis a statistically significant (p=0.099) relationship between thepresence of the 11 repeat allele and the proportion of panellists withvisual skin dryness (n=113).

FIG. 7 shows the proportion of panellists with or without the 12 repeatallele who displayed decreased erythema between 4 and 48 hours postpatch with 1% SLS. Fisher's Exact test: There is a statisticallysignificant relationship (p=0.0587) between the presence of the 12repeat allele and decreased erythema post patch (n=131).

EXAMPLES Methods

DNA isolation was performed using the QIAamp DNA Blood Midi Kit (Qiagen)and the method was adapted for saliva samples as described by Schie andWilson (1997, Journal of Immunological Methods, 208, 91-101). 5 ml ofsaliva was diluted with 5 ml PBS (Sigma) and centrifuged for 5 mins at3000 g. The supernatant was discarded and the pellet resuspended in 10ml PBS. The resuspended pellet was centrifuged for 5 mins at 3000 g, thesupernatant was discarded and the pellet resuspended in 750 μl PBS. 1 mlkit Lysis buffer and 100 ul kit protease was added according to the kitprotocol. 5 μl RNase (7 units/μl) was added followed by mixing andincubation at 70° C. for 20 mins. 1 ml absolute ethanol was added andthe kit protocol was followed to purify the DNA. The final preparationwas eluted in 300 ul kit elution buffer, and the eluate was re-appliedto the column once in order to concentrate the preparation. The eluatewas stored in aliquots at −20° C.

DNA quantitation was performed on a 1/10 dilution by spectrophotometricmeasurements (Sambrook and Russell, (2001) Molecular Cloning: ALaboratory Manual. 3rd edition, Cold Spring Harbour Laboratory Press,New York, USA).

PCR reactions were performed in a 50 μl volume using the followingconditions: 150 ng Genomic DNA sample; 200 μM final concentration dNTPs;200 μM final concentration forward (SEQ ID NO: 1: 5′ GGA TGA AGC CTA TGACACCAC 3′) and reverse (SEQ ID NO: 2: 5′ GA CAG GAA AAG ATA ACT TCC C3′) primers; 1.7 mM final concentration Mg²⁺. For 50 μl Elongase(GIBCOBRL Life Technologies) reaction use 3 ul buffer A+7 ul buffer B.The PCR reaction was performed using the following conditions:

Heat reactions to 94° C. for 5 minsAdd Elongase reaction mix to 1/50 dilution (HOT START)Start programme:

 5 minutes 94° C.  1 cycle 30 seconds 94° C. 30 seconds 57° C. {closeoversize brace} 35 cycles 12 minutes 68° C. soak  4° C.

PCR products were analysed by gel electrophoresis using techniques wellknown in the art (Sambrook and Russell, (2001) Molecular Cloning: ALaboratory Manual. 3rd edition, Cold Spring Harbour Laboratory Press,New York, USA). Sample loading buffer GIBCO-BRL was added to an aliquotof the PCR product to a 1× final concentration. 20 μl of PCR reactionproduct was size fractionated on a 0.6% agarose (1×TBE Sigma) gel byelectrophoresis at 30V (using extension ladder markers—LifeTechnologies).

Example 1

A group of 43 randomly selected females provided saliva samples for DNAanalysis, and information on skin condition history. Panellists weresubjected to a patch of 1% SLS (sodium dodecyl sulfate) for 24 hours andthe degree of erythema was scored 4, 24 and 48 hours after the patching.In a separate study a randomly selected mixed gender group of 20individuals provided saliva samples for DNA analysis, cyanoacrylatebiposies (Marks (1972) British journal of Dermatology 86:20-26) for NMFanalysis and a skin/scalp condition history. Genomic DNA was extractedfrom the saliva samples and analysed by PCR to determine theprofilaggrin genotype for each individual (FIG. 1). The NMF content ofthe stratum corneum was determined using the standard TNBS assay (Hazraet al 1984 Analytical Biochemistry 137: 437-443) and normalised to totalprotein.

All three variants were identified with the following frequencies:

10 repeats (18%), 11 repeats (60%), and 12 repeats (220).

A plot of filaggrin repeat number per chromosome pair versus stratumcorneum NMF levels revealed a direct relationship between these twoparameters (FIG. 2). Individuals with higher repeat numbers had higherNMF levels. The skin/scalp condition history revealed that individualswho claimed to have suffered from dandruff carried 1 or more copies ofthe 12 repeat allele (FIG. 3). These results suggest an associationbetween the presence of the 12 repeat allele in the genome of anindividual and susceptibility to dandruff.

A plot of erythemal score after SLS patch (FIG. 4) revealed thatindividuals carrying 1 or more 12 repeat alleles had lower erythemalscores 48 hours after challenge. These results suggest an associationbetween the presence of the 12 repeat allele and recovery of the skinbarrier after detergent challenge.

Example 2

A group of 140 randomly selected panellists provided saliva samples forDNA analysis, and information on skin condition history. On the basis ofresponses to the questionnaire panellists were assigned self-perceiveddry skin frequency—‘Frequent’ (including always, daily and weekly) and‘Infrequent’ (monthly or less often). Visual assessments of thecondition of panellists untreated leg skin were made on 5 consecutivedays and panellists grouped as ‘No skin dryness’ (3 or more assessmentswith no visual dryness) or ‘Skin dryness’ (more than 2 assessments withvisual dryness). Panellists were subjected to a patch of 1% SLS for 24hours and the degree of erythema was scored at 4, 24 and 48 hours afterthe patching. Genomic DNA was extracted from the saliva samples andanalysed by PCR to determine the profilaggrin genotype for eachindividual (FIG. 1).

All three variants were identified with the following frequencies:

10 repeats (24%), 11 repeats (56%), and 12 repeats (20%).

The proportion of panellists of panellists with at least one 12 repeatallele who claimed to suffer from frequent dry skin was only 6% comparedwith 27% of panellists with no 12 repeat alleles (FIG. 5). Thisdemonstrated that the 12 repeat allele is associated with a reducedtendency to report self-perceived dry skin.

The proportion of panellists with at least one 11 repeat allele whodisplayed visual leg dryness was 73% compared with only 52% ofpanellists with no 11 repeat allele (FIG. 6). This demonstrated that the11 repeat allele is associated with an increased tendency to visual legdryness.

The proportion of panellists with at least one 12 repeat allele whoshowed a decrease in erythema between 4 and 48 hours post SLS patch was66% compared with only 47% of panellists with no 12 repeat alleles (FIG.7). This again demonstrated that the 12 repeat allele is associated withan increased tendency of the skin to recover after detergent challenge.

1-25. (canceled)
 26. A kit for determining whether predisposition of anindividual to a skin condition selected from the group consisting ofdandruff, dry skin and detergent induced erythema correlates with theindividuals level of profillagrin alleles present in the genome, saidkit comprising oligonucleotide primers which can be annealed tosequences flanking the polymorphism and within or surrounding theprofilaggrin gene in order to prime amplifying genomic DNA of a sampletaken from the individual that correspond to profillaggrin alleles ofdifferent filaggrin repeats.
 27. A diagnostic kit according to claim 1wherein the primers are designed to distinguish alleles having 10 or 11or 12 filaggrin repeats.
 28. A diagnostic kit according to claim 2, thekit comprising: (a) A forward polymerase chain reaction primer, theforward primer having a nucleotide sequence SEQ ID NO: 1: 5′ GGA TGA AGCCTA TGA CAC CAC 3′; and (b) A reverse polymerase chain reaction primerhaving a nucleotide sequence SEQ ID NO: 2: 5′ GA CAG GAA AAG ATA TCC C3′.
 29. The kit according to claim 26 further comprising DNA polymerase.30. The kit according to claim 26 further comprising dNTPs suitable fordilution to a 200 μM final concentration, a pair of oligonucleotideprimers SEQ ID NO: 1: 5′ GGA TGA AGC CTA TGA CACCAC 3′ and SEQ ID NO: 2:5′ GA CAG GAA AAG ATA ACT TCC C 3′, magnesium chloride solution suitablefor dilution to 1.7 mM final concentration Mg²⁺ and Elongase.
 31. Thekit according to claim 26 further comprising a personal care productwhich is suitable for use on an individual having a particularprofilaggrin genotype.
 32. The kit according to claim 26 furthercomprise instructions for using the components.